What is HPLC and how does it work?

User HPLC

HPLC stands for High Performance Liquid Chromatography, formerly referred to as High-Pressure Liquid Chromatography.

It is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component. In general, the method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. for the separation, identification, and quantification of the sample mixture.

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The term chromatography

The term chromatography comes from the Greek word chroma- which means color and -graphein which means writing. The first recorded use of column chromatography is given to the Russian scientist Mikhail Tsvet who crushed calcium carbonate in a tube and subsequently added homogenized leaves of a green plant, followed by an organic solvent. Tsvet observed separate colored bands as the solvent passed through the tube. This is how practical chromatography began at the very beginning, successfully separating various pigments from the leaves. In today's world, there are many analytes that are colorless and separated by chromatographic techniques, it is still known by the same name.

High Performance Liquid Chromatography (HPLC)

It is a type of column chromatography in which by the action of a pump, a mixture of compounds or analytes is passed in a solvent system commonly known as mobile phase. The mobile phase passes through a chromatographic column, which contains the stationary phase at a specified flow. The separation of the compounds occurs on the basis of the interaction of these with the mobile phase and the stationary phase.

Ultra-High Performance liquid chromatography (UHPLC)

It is a technology based on the principle that a smaller particle size leads to greater efficiency, rapid separations with higher resolution and sensitivity. However, in order to withstand the extreme pressure of particles smaller than 2 µm, the equipment needs to be able to work at high back pressure. The efficiency of these columns should not be lost on the rest of the equipment, due to the dead volume, that is why equipment capable of working at high pressures was designed.

Due to the high cost of UHPLC instruments, at the end of 2000, Phenomenex launched the Kinetex 2.6 µm columns with Core-Shell technology, which provides UHPLC performance on traditional HPLC equipment. In this way, the traditional HPLC instrument that is owned in the laboratory can be used and achieve an equivalent UHPLC performance.

Modes of separation.

There are many modes of separation in chromatography and each has its own principles.

The following is an HPLC column selection guide to help readers choose the correct analysis mode. Although there are many separation modes available to resolve the mixtures in a chromatograph, Reverse Phase separation (RP) is the most common mode in liquid chromatography.

“Why is the reverse phase called the reverse phase?”

The answer is simple:

Chromatography evolved from the use of the polar stationary phase and a non-polar eluent as the main component of the mobile phase, so it was considered the normal practice, hence the name normal phase. While this model separated components based on their polar nature, there were a large number of analyte mixtures that were non-polar and had hydrophobic characteristics that needed separation.

The use of a non-polar stationary phase, with a polar mobile phase helped to separate these hydrophobic analytes. Since this practice is inverse to the normal phase, the term reverse phase is used. This is similar to calling a right-handed ping-pong player as normal and a left-handed ping-pong player as the original.

Reverse phase separation process

Now that we know that the most popular mode of liquid chromatography is the reverse phase, let's explore how it works.

Below is a schematic of the separation process. The analyte mixture is represented by blue, purple and red dots, which are introduced together into the column containing a non-polar reverse phase stationary phase. The red arrows represent the direction of the flow of the mobile phase. When the mixture of mixed analytes enters the column, the mobile phase pushes the analytes down the column. As they move forward they come into contact with the stationary phase. Analytes that have a higher affinity for the stationary phase (blue dots) will be retained more strongly and will elute later in the run. Therefore, you can separate the analytes based on the intensity with which they interact with the stationary phase.

In the following example a stationary, hydrophobic non-polar phase is represented, more specifically a C18. The mobile phase consists of a polar, hydrophilic aqueous component, usually water and acetonitrile or methanol. The analytes will be separated based on their relative affinity for these two phases. Hydrophobic compounds, such as benzopyrene, will have a strong affinity for the hydrophobic stationary phase, and will be strongly bound. Hydrophilic compounds such as ethyl sulfate will have little affinity for the stationary phase and will remain mainly in the mobile phase and will be transported rapidly through the column.

Although reverse phase separation occurs by hydrophobic interaction, there are three primary mechanisms of interaction that dictate the overall chromatographic behavior.

This includes:

Hydrophobic interactions
Polar interactions

Ionic interactions

Apart from these three interactions, steric selectivity and the shape of the molecule can sometimes also contribute to the interaction. Using the example of tapentadol, a typical pharmaceutical molecule of small size, we can better show these three types of interaction, since it has polar, hydrophobic and also ionic components.

Hydrophobic Interactions

It is the primary mechanism in RP-HPLC and that determines the retention behavior. That is the hydrophobic interaction between the non-polar stationary phase ligand (e.g. C18) and the hydrophobic nature of the sample molecule (e.g. the carbon skeleton).

This is a weak and transient interaction between a non-polar stationary phase and molecules, which includes hydrophobic and van Der Waals interactions. A reasonable estimate of retention can be predicted based on the Log P value, or octanol-water partition coefficient coefficient. The Log P is the ratio of octanol to water in a liquid-liquid extraction. In other words, the more hydrophobic a molecule is, the higher the Log P value it has, which translates into higher retention in RP-HPLC.

Polar Interactions

These are interactions that occur between the polar functional groups of analytes. They also occur among residual silanols, embedded polar groups, surface polar groups or polar end groups in the stationary phase. They interact with the analyte through hydrogen bonds and dipole-dipole. These interactions are relatively weak and transient compared to the ion exchange interaction.

Ion Exchange Interactions

Most of the stationary phases in RP are based on a silica support to which a non-polar phase such as C18 is anchored. Chromatographic column manufacturers such as Phenomenex, try to achieve the complete inactivation of all silanol groups of silica, however this process cannot eliminate 100% of the groups, giving rise to residual silanol groups on the surface (Si-OH) that are hidden. These silanols can deprotonate and acquire a negative charge, and ionically interact with positively charged basic analyte molecules. These ion exchange interactions are very strong and slow, in contrast to hydrophobic and polar interactions. Therefore, when ion exchange occurs, analytes experience different rates of interaction (slow vs fast), and this can lead to peak distortion. This is a classic example of basic analytes interacting with residual silanols, which can be controlled by neutralizing the silanol or neutralizing the analyte by analyzing them at high pH.

¿How does work?

In very small amounts, the sample mixture to be separated and tested is sent into a stream of mobile phase percolating via a column. There are different types of columns available with sorbents of varying particle sizes and surfaces. The mixture moves through the column at varying velocities and interacts with the sorbent, also known as the stationary phase.

The velocity of each component in the mixture depends on:

Its chemical nature.

The nature of the column.
The composition of the mobile phase.

The time at which a specific analyte emerges from the column is termed as its retention time. The retention time is measured under specific conditions and considered as the identifying characteristic of a given analyte.

Sorbent particles might be hydrophobic or polar in nature. The commonly used mobile phases include any miscible combination of water and organic solvents such as acetonitrile and methanol. Water-free mobile phases can also be used.

Differents types of HPLC

The following are the types of HPLC based on the stationary phase in the process:

Normal Phase HPLC

NP HPLC separates the molecules according to polarity, in which the polar stationary phase and the non-polar mobile phase is used.

Reverse Phase HPLC

The reverse phase chromatography works on the principle of hydrophobic interactions so the more nonpolar the analyte has, the longer it will be retained. It this mobile phase is polar and the stationary phase is nonpolar in nature.

Size Exclusion HPLC

Size Exclusion Chromatography (SEC) is a chromatographic process that separates molecules based solely on their size, in this technique molecules are separated by the column packing material on the basis of their exclusion from pores.

Ion Exchange HPLC

It uses to separate the ions and polar molecules based on their affinity to the ion exchanger. Ion exchange chromatography is the most popular method for the purification of proteins and other charged molecules.

Experimental procedure

Wash absolutely the whole chromatographic system, as a prevention, with washing solvent, for example H2O:ACN (70:30) for a time not less than one hour.
Install column (C8 / C18) properly as required.
Prepare the mobile phase and fill it in the reservoir. (Solvent, Buffer, or combination of it).
Purge the mobile phase reservoir by opening the purging valve.
Gradually increase system flow up to the required flow rate, and wait until the column is saturated and the baseline is corrected.
Adapt the chromatographic system to the mobile phase for at least thirty minutes.
Prepare samples in different concentrations, as needed.
Load or build a method, and fill parameters such as flow rate, the mobile phase composition, wavelength, oven temperature, and program time.
Create a sequence for samples and save it.
As you get a baseline, then inject the sample manually or by auto-sampler via injector (sample loop/syringe).
After analyzing the sample, study the retention time, tailing factor, capacity factor, and theoretical plates of each peak.
Repeat the process according to the number of samples.
Wash the column properly by HPLC grade water, and methanol/ acetonitrile.

HPLC Applications

Advantages of HPLC are as follows

The high performance liquid chromatography provides a simple, automated, and highly accurate method of identifying certain chemical components in a sample.
Provides a quantitative and qualitative analysis that is simple and accurate.
It can be upgrading to mass spectroscopy.
Compared to other chromatographic techniques such as column chromatography, TLC, and paper chromatography, HPLC is fast, effective and delivers high resolution.
The gradient elution is readily adaptable in HPLC.

Disadvantages of HPLC are as follows

This requires a large number of expensive solvents, power supplies, and regular maintenance.
Need to be expertise, since it is more difficult for beginners.
The reliability of the separation process depends on the cleanliness of the mobile phase, sample and proper system operation.
The contaminated column can affect the peak shapes.

¿What kind of precautions we need to take during analysis?

Make sure the column washed before and after analysis.
Solvents must be filtered through a 0.5 μm nylon filter membrane and degassed.
The sample must be particle-free, therefore filtered through a 0.2 μm nylon filter membrane.
Buffers like phosphate buffers, acetate buffers, etc. are very harmful to the HPLC system and columns they need to be washed properly.
Don’t overload the column.
Use appropriate flow rates to maintain system pressure.
Do not run HPLC systems at high backpressure.
Always use grade solvents and water derived from reliable sources.
Use guard columns to protect against contamination and prolong column life.
Don’t use a mobile phase or buffers with a highly acidic or basic pH.